Clostridium difficile colitis (CDC) is a toxin-mediated inflammatory lesion of the colonic mucosa usually induced by administration of antibiotics or cancer chemotherapeutic agents. By discrupting the normal microbial flora of the colon, such agents permit C. difficile to reach a large population level. The goal of this research project is to determine the biochemical and physiologic mechanisms by which the normal flora suppresses C. difficile and to identify species of bacterial responsible for this suppressive effect. An anaerobic continuous flow (CF) culture system developed by Dr. Rolf Freter will be used to simulate the ecosystem within the cecum of the Syrian hamster. This animal has already proven to be a very useful model for CDC. The CF culture system to be used has been shown to reproduce in vitro an ecosystem with the composition and functions of the complex microbial community of the normal rodent cecum. Use of the CF culture colonized with the normal hamster cecal flora will allow manipulation of physicochemical conditions in vitro. Study of the effect of these manipulations on the population size of C. difficile will give insights into the mechanisms by which the normal flora suppresses C. difficile in vivo. In addition, microorganisms will be isolated from the normal cecal flora in order to develop a simplified, defined flora able to exert a potent antagonistic effect against C. difficile. Data from CF cultures, intact hamsters and genotobiotic mice will be compared to test the validity of data from the in vitro system. Then, CF cultures will be used to study mechanisms by which the human colonic flora suppresses C. difficile and to develop a simplified, defined human flora able to antagonize this pathogen. These studies are intended to lay some of the groundwork needed for clinical manipulation of the human gut flora to suppress colonization by pathogens.